Sterilization of collagenous sutures with epoxides



Feb. 23, 1960 w. L. GEORGE ETAL Re' 24337 STERILIZATION oF coLLAGENoussuTuREs WITH EPoxIDs original Filed Jan. 15. 1957 ATTORNW ilnited StatesPatent lice Re. 24,787 Reissues Feb. V23, 1960 STERILIZATION OFCOLLAGENOUS SUTURES WITH EPOXIDES William L. George, New Brunswick, NJ.,and James J. Eberl, Moylan, Pa., assignors to Johnson & Johnson, acorporation of New Jersey Original No. 2,817,437, dated December 24,1957, Serial No. 634,353, January 15, 1957. Application for reissueDecember 23, 1958, Serial No. 782,614

24 Claims. (Cl. 206--63.3)

Matter enclosed in heavy brackets I] appears in the original patent hutforms no part of this reissue specication; matter printed in italicsindicates the additions made by reissue.

This application is a continuation in part of Serial No. 302,004, nowabandoned.

This invention relates to sterilization of proteinaceous materials withepoxide sterilizing agents, especially surgical sutures.

The well-known catgut sutures made from sheep or other animalintestinesare proteinaceous or collagenous in composition and hence aremarkedly affected by water, and to' a much greater extent by combinedheat and water. Excessive amounts of water tend to swell the suture,which is undesirable, while heat and water together hydrolyze thecollagen thereby weakening the suture.

rIlle suture itself must, of course, be sterile when it is used and, inaddition, must be packaged in a receptacle which, at the time it iscarried into the operating room is sterile also. However, heat or steamsterilization procedures ordinarily used for other articles are notcompatible with the sensitivity of catgut to these conditions asdescribed above. Chemical sterilizing agents have been proposed. Asatisfactory chemical sterilizing process should not unduly swell thesuture and should not cause appreciable decrease in tensile strength.Absorbability of the suture by body tissues should not be lessened. Manyof such processes are too rigorous in their action and even the milderones heretofore known produce deleterious effects on the more sensitivearticles such as collagenous sutures.

An object of the invention is to develop a method for sterilizingarticles, particularly sutures made from animal intestines, which do'esnot cause deterioration of the article being sterilized, and which doesnot materially dei crease absorbability of the animal intestine sutures.A further object is to discover a way of modifying and controllingepoxide sterilization procedure to make it applicable to treatment ofless refractory materials. Other objects and advantages of the inventionmethod will aph pear hereinafter.

According to the invention, articles are sterilized by treating themwith liquid epoxide in certain concentration, in the presence of acontrolled amount of moisture.

A preferred modification of the invention is based on the discovery thatcertain co'mpounds, termed herein as modifiers, when added to liquidepoxide sterilizing agents permit sterilization of articles withoutdeleterious action toward the article being sterilized which mightotherwise' occur; i.e., by adding to the mixture one-of the inventionmodifiers which are ammonium salts o'f the lower molecular weight (8carbon atoms or less) aliphatic solublehydroxy carboxylic acids. Theamount of modifier and ratio of ammonia to acid therein are preferablysuch as to produce a controlled pH in the mixture as described morefully below. Further the temperature is at a level which will avoiddegradation of the article, and the sterilizing conditions aremaintained for time to effect sterility.

In the description of the invention reference will be made to theaccompanying drawing, which shows a tubed suture, the tubing fluid ofwhich includes epoxide sterilizing agent.

Referring to Figure I of the accompanying drawing, a proteinaceous orcatgut suture 11 (arranged in any desired fashion) is enclosed in a tube10 (hermetically sealed), and the tubing uid 12 completely covers thesuture. This tubing liuid contains the epoxide sterilizing agent.

Sterilization according to the invention procedure, as applied tosterilization of sutures, is carried out as follows. The catgu suture tobe sterilized is preferably first dried, as for example with warm air.Its moisture content is preferably reduced to below about 8% by weight.The suture then may be Wound upon a suitable spool, the spool and sutureinserted into the top of a closed-bottom glass tube and the tubing fluidand epoxide sterilizing agent added to the tube so as to cover thesuture. Preferred tubing liuids are isopropyl alcohol or ethyl alcohol.However,any iiuid which will dissolve the required amounts of water,epoxide and modifier may be employed. The fluid should also benondeleterious tok the suture.

The ethylene oxide or propylene oxide are volatile and hence are usuallyhandled in the form of solutions that are either aqueous or dissolved intheV tubing uid itself. If desired, however, the epoxde may bemaintained under pressure to prevent excessive volatilization. Epoxideoontent is suicient to produce sterility, generally at least 0.01%,desirably at least 0.25% by weight of the duid, and preferably at leastabout 0.50%. Concentrations above 2.5% may cause undesirable effects inthe article sterilized, and hence are usually avoided. The volumepercentage is about 1.1 times the percentage by weight.

To promote activity of the epoxide sterilizing agent, the presence ofcertain minimum amounts of water is important. We have found in theinvention process that the minimum amount of water for reliable andconsistent resultsv in sterilization is about 21/% by weight. However,inthe case of sutures, water also affects the pliability (brittleness)and swelling thereof. With isopropyl alcohol tubing liuid, water contentof the` fluid should be maintained at least about 5% by weight` topreserve pliability Iof the sutures. Above about 20% water in isopropylalcohol, undue swelling of the sutures has been found to occur. Hencethe broad range of water content 'in isopropyl alcohol is 5 to 20%.Preferred iso' propyl alcohol tubing fluids contain 8 to 12% water. Inthe case of ethyl alcohol tubing fluids, similar rules apply but thebroad range of water content is about 2 to 10% and the preferred range 4to 6%.

An outstanding feature of the invention method is using a sterilizingagent modifier, whereby the articles are sterilized without deleteriouseffect thereon. The modifiers contemplated are ammonium salts of thelower molecular Weight aliphatic carboxylic acids. The amount of modierand ratio of ammonia to acid therein are preferably controlled to'produce in the final uid mixture a pH within certain ranges. Ammoniumlactate is a particular and preferred modifier in the invention method.It is suitably incorporated into the uid by first adding the requiredamount of lactic acid, at least 0.25% by weight, and then addingammonium hydroxide until the pH has been raised to the desired level.The pH range is about 5.0 to about 8.5. ln the case of ethylene oxidesterilizing agent, the preferred pH range is abo'ut 5.0 to about 7.0.According to the invention method, amounts of ammonium lactatecorresponding to not more than about 5% lactic acid, and pHs of about5.0 to about 7.0 for ethylene oxide and 5.0 to' 8.5 for propylene Oxideeffect sterilization with substantially no deterioration in properties.Since certain of the modifiers have a plasticizing and swelling effecton sutures, fluids used for suture sterilization will generally containnot more than 2.5 moditier, preferably not mOre than about 1.0%.

After adding solvent, water, epoxide, and a modifier to the suture orother article in the tube, the tube is sealed olf and stored at normalroom temperature conditions for time suicient to bring aboutsterilization of the suture. In this period the epoxide is substantiallydecomposed. 'I'he time required will depend in part upon the temperaturelevel maintained. In general, temperatures above about 50 F., are used.Above about 100 F., deterioration of the suture may take place, andaccordingly such higher temperatures are avoided. Normal roomtemperatures are suitable and hence are preferred, at which temperaturesubstantially complete disappearance of the epoxide and completesterilization of the suture will take place in about ten days to twoweeks.

It is animportant advantage of the invention method 'as indicated abovethat the sutures become sterilized without undergoing appreciableswelling or reduction in tensile strength, and that absorbability is notmaterially affected thereby.

The invention method has been `described mainly as applied to catgutsutures. However, it will be apparent that the method is also applicableto sutures other than those prepared from animal intestines and, broadlyconsidered, other materials which are adversely affected bysterilization with liquid epoxides.

The following examples are submitted as illustrations of the invention.The examples do not, however, limit the invention since otherembodiments not illustrated come within its scope. Although the languageof the examples describes a single test, the results reported representconsistent data from a large number of experiments.

EXAMPLE I A catgut" suture, prepared from sheep intestines, and havingan original diameter of about 0.022" and about 12% moisture content, iswound upon a spool and placed in a vertical tube having its bottom yendsealed and top end open. The suture is contaminated by adding to it asuspension of B. subtilis and drying. A tubing nid consisting of 90% byweight isopropyl alcohol, 1% ethylene oxide, 1% lactic acid withsuicient ammonium hydroxide to increase the pH to 6.5, and the balancewater, is then added to the tube so as to cover the suture therein. Thetubing fluid is prepared as follows: 700 ccs. of 99% isopropyl alcoholis mixed with 14 ccs. of 50% aqueous lactic acid solution and 49 ccs. ofwater. 28% aqueous ammonium hydroxide is then added until the pH isincreased to 6.5. To 130 ccs. of this stock solution there is added 1.80ccs. of ethylene oxide (to produce a 1% by weight ethylene oxidecontent). After adding the tubing fluid, the tubes are sealed and thesutures allowed to stand at room conditions Afor ten days. The sampletubes are then opened and portions of the tube contents transferred to athioglycolate medium using aseptic technique. After a fifteen dayincubation period, the sutures are examined and all found to be sterile.Control tests run in a manner identical with those described above,except that no ethylene oxidel is used, are all non-sterile.

Some of the samples are subjected to aging tests for one year at roomtemperature. At the end of this time the tubes are opened and the sutureis tested for swelling and tensile strength. They are found to haveundergone substantially no swelling and to have suffered substantiallyno decrease in tensile strength.

In tests similar to Example I wherein no ammonium lactate is added tothe tubing fluid, there will be found, upon aging, a reduction intensile strength of about 50% and increase in diameter due to swellingof about %.l

4 EXAMPLE u Example I is repeated except that propylene oxide is used inplace of ethylene oxide and the pH is adjusted to 8.5, using ammoniumhydroxide, instead of 6.5 as in Example I. After being stored for thirtydays at room temperature, the tubes are opened and found to be sterile.Parallel control of tests without propylene oxide are found non-sterile.'Ihe samples are subjected to aging tests and examined after storage forone year at room temperature. The sutures are removed from the tubes andtested for swelling and tensile strength. They are found to haveundergone no substantial decrease in tensile strength and no substantialincrease in diameter as compared with the properties of the originalunsterilized suture.

Tests in which the Example II procedure is repeated except that noammonium lactate is added, show substantial decrease in tensile strength(50% of the original) and substantial increase in diameter (10% of theoriginal) due to swelling upon being subjected to aging tests by storageat room temperature for one year.

EXAMPLE III The procedure of Example I is repeated except that 2.5%lactic acid is added to the tubing fluid and the pH is adjusted to 5.5with ammonium hydroxide. At the end of 12 months storage time at normaltemperature conditions, the suture `diameter measures 0.0226", the knotpull strength is substantially greater than the minimum required byU.S.P., and there is no trace of ethylene oxide in the tubing fluid.

As a result of a series of in-vivo tissue implantation studies, it wasfound that catgut sutures sterilized by this new process were equally asabsorbable as sutures sterilized in the usual manner. However, in thecase of the ethylene oxide or propylene oxide sterilized catgut there isan indication that attack is slower at first increasing with time to apoint Where it finally completely absorbs in about the same length oftime as cumolized gut.

Thus the ethylene oxide or propylene oxide sterilized gut has theadvantage of retaining a greater strength when the Wound is weakest andsuture strength is most needed. This is a completely unpredictableresult.

The materials of the foregoing examples give acceptable values whentested for digestion in a papain solution, e.g., according to thefollowing procedure:

Eight-inch lengths of the material to be tested are placed side by sideforming a loop of the material, and the two cut ends are treated as onedouble strand. A single overhand throw-knot is tied approximately 1A ofan inch from the cut ends, thus forming a loop about 3% inches long.These samples may be placed in glassine envelopes, and may be storedtherein for several days if necessary.

The digestion is carried out in tubes 1 inch in diameter and 4 incheslong. These are placed Yin a constant temperature bath at 37.8 C. (100F.). Twenty grams tension is placed on the loop, eg., by means of alever and weight arrangement. The digestion (breaking of the gut) timemay be measured manually. Alternatively, a mercury switch may befastened to the lever so that when the loop is intact and submerged inthe digestion solution, current ows through an electric timer. When theloop breaks, the lever with the twenty gram weight changes position,switching off the current. Only the time during which the loop wassubjected to digestion is recorded on-the timer.

The digestion solution comprises:

Buffer solution (1.0 liter) Potassium phosphate-dibasic KRHPO; anhydrousl g 174.22 Sodium borate Na2B4Oq-*10H2O g 38.15 Distilled water ce..`1000 the mortar which is mixed to form a heavy paste.

Papain solution (1.0 liter) Buffer solution (above) cc 100 ThioureaCS(NH2)2 g 76 Distilled Water cc 800 Powder papain (optimo white brandS. B. Penick &

Company) g 30 and may be prepared in a stainless steel beaker using anelectric stirrer with a stainless propeller and shaft. The water and thebuffer solution are poured into the stainless steel beaker and the speedof the stirrer is adjusted for vigorous mixing without splashing. Thebuffer and water are allowed to mix while the thiourea is being weighedout. This is then poured carefully into the stirring mixture and allowedto dissolve while the papain powder is being' weighed out. The weighedpapain powder is then placed into a large mortar. When the thiourea hasdissolved, a quantity of the solution (from 20G-30() cc.) is dipped outwith a beaker. A small amount of 'this solution is added to the papainpowder in The heavy paste is rubbed, until all the lumps have beenbroken; then more of the solution from the beaker is added until a thinpaste forms. This is transferred to the stainless steel beaker. Theremainder of the dipped solution is used to rinse the mortar into thestainless steel beaker. Mixing is continued with the stirrer, preferablyfor about at least 5 hours, and the solution is allowed to stand coveredovernight at room temperature. Standing for a weekend has no adverseeffect on activity.

The resulting solution is filtered, the liquid layer being carefullypoured off into another container without disturbing the sediment. Afilter aid (such as Filter Cel) is mixed into the liquid (about 5teaspoons per liter of solution) and the mixture is allowed to standwhile vacuum `ltration apparatus is set up. The filtration apparatusconsists of a filtering ask (4 liter) and a Buchner funnel (about 20'cm. in diameter). A disk of filter paper (Whatman #1) is placed in thefunnel, wet with distilled Water and pressed flat, covering all theholes'. The mixture of papain solution and filter aid is stirred andimmediately poured into the funnel in such a manner as not to disturbthefilter paper. If the filtrate cornes through cloudy, it is poured backand refiltered until it comes through clear. When the liquid fractionhas been iiltered, the sediment from the first step is filtered. Theresulting filter cake is then washed with 50` cc. of distilled water perliter of palpain solution. The filtrate is measured and diluted to thedesired volurne with distilled water. This solution should be usedwithin a period of two weeks, after which decreasing activity of thesolution makes it useless.

Just before use, the above solution is mixed with an activator made upof100 cc. of distilled water containing 4.75 grams of meta sodiumbisulfite `(Na2S2O5) or 5.2 grams of sodium bisuhitev(Nal-ISO3) whichchemical dissolves easily in the water, in the following proportions(per sample of testrmaterial):

Papain solution cc 24.0 Activator cc 1.0

Since the activity of the papain solution is variable, it is necessaryto have a method of standardization. For this, a size-0, unbleached,non-hardened (i.e., nontanned)nonboilable gut is used, and it shoulddigest in 1.8 hours.

At least three such standard digestions should be run with each batch oftest digestions. lf the standard digests in 1.8 hours, direct test batchtime readings may be made. If there is adierence, Le.,v 1.5 for thestandard, a conversion factor must be used to get the proper digestiontime for the material. This factor is found by dividing 1.8 by the timeof digestion of the standard;

in this case 1.8/ l.5'=1.20. All readings in this c's must be multipliedby the factor (in this case 1.20) to obtain the correct values.

If all of the advantages of the foregoing modifications of the inventionare not required, the sterile sutures which meet minimum U.S.P. tensilestrength requirements and having acceptable papain digestioncharacteristics may be prepared as follows:

EXAMPLE'IV Lbs.

Average 20.6 lbs. (U.S.P. minimum for No. 2 catgut; 13 lbs.). Thisclearly shows that the sterile sutures prepared in accordance with thismodification of the invention clearly meet minimum U.S.P. tensilestrength requirements.

vThe papain digestion breaking time for tive of the sterile sutures(using the above described procedure) is:

Hours Average These data clearly show that the sterile sutures preparedin accordance with this modification of the invention are clearly withinacceptable absorption requirements; ire., at least 3 hours desirably atleast 6 hours, and preferably at least 11 hours.

EXAMPLE V A number 2A plain gut suture is tubed by the procedure ofExample I in a solution of 2% ethylene oxide by volume in 87%isopropanol (aqueous). inoculated tubes are allowed to stand at roomconditions for one week. The resulting sutures areI subjected tostandard sterility tests and all found to be sterile, whereas thecorresponding control tubes (no ethylene oxide added) are all found tobe non-sterile. The tensile strength for five of the sterile sutures isas follows:

. Lbs. 23.6

4 Average 21.4 lbs. (U.S.P. minimum for No. 2 catgut: 13 lbs.). Thisclearly shows that the lsterile sutures prepared in accordance with thismodification of the invention clearly meet minimum U.S.P. tensilestrength requirements.

The sealed The papain digestion breaking time for ve of the sterilesutures (using the Vabovedescribed procedure) is:

f Comparable results to the foregoing are achieved using variousmodifications thereof. For instance, with a number 2A plain gut, and a1.33% by weight solution of ethylene oxide in 90% isopropanol (aqueous),tensile strength is about 25, the papain digestion average of iive tubesis 11.25 hours (range 6.0 to 23.0), and the reduction in shrinkagetemperature is about 6 C. ln a similar test except using 0.5% ethyleneoxide, the tensile strength is about 27.65 lbs., the papain digestiontime is 11.88 hours average (range 9.3 to 18.7) and the decrease inshrinkage temperature is about 2 C. Other suture materials may betreated in accordance with this invention as descrdibed hereinabove,including tanned gut, chromic gut, and the like.

In view of the foregoing disclosures, variations or modificationsthereof will be apparent, and it is intended to include within theinvention all such variations and modilications except as do not comewithin the scope of the appended claims.

We claim:

1. A method of sterilizing collagenous suture material by immersing itin a liquid comprising about 2.5 to 20% of water based on the weight ofthe liquid, a sterilizing epoxide of the group consisting of ethyleneoxide and propylene oxide in an amount of the range of about 0.25 to2.5%, for a time in the range of about to 14 days and va temperature inthe range of about 50 to 100 F., the improvement which comprisescarrying out said method in the presence of an ammonium salt of ahydroxy carboxylic acid having not more than 8 carbon atoms in an amountto provide a pH in the range of about 5.0 up to about 7.0 in the case ofethylene oxide and up to about 8.5 in the case of propylene oxide, and asolvent for said salt water and epoxide which is nondeleterious to saidcollagenous material, whereby said collagenous suture material issterilized without causing substantial deterioration thereof.

2. A method of claim l wherein the hydroxy carboxylic acid is lacticacid.

3. A method of claim 2 wherein the lactic acid salt is present in anamount equivalent to 0.25 to 5.0% lactic acid.

4. A method of claim 1 wherein the suture material is prepared fromanimal intestines.

5. A methodof claim 4 wherein the hydroxyy carboxylic acid is lacticacid and the salt thereof is present in an amount equivalent to 0.25 to2.5% lactic acid.

6. A method of' claim` 5 carried out in a sealed tube wherein theepoxidc is ethylene oxide, the equivalent amount of lactic acid is 0.25to 1%, and pH is in the range of 5.0 to 7.0.

7. A method of claim 6 wherein the solvent is isopropyl alcohol.

8. A method of claim 7 wherein the amount of lactic acid is 1%.

9. A method of claim 8 wherein the suture material is dried to amoisture content not substantially above about 8% by weight prior toimmersion in the liquid, and wherein the liquid mixture contains 8 to12% water.

l0. A method of claim 5 wherein the solvent is ethyl alcohol.

11. A method of claim 4 carried out in a sealed tube wherein the epoxideis -propylene oxide, the equivalent 8 amount of lactic acid is 0.25 to1%, and the pH is in the range of 5.0 to 8.5.

l2. A method of claim 1l wherein the solvent is isopropyl alcohol.

13. A method of claim 12 wherein the amount of lactic acid is 1%.

` 14. A method of claim 13 wherein the suture material is dried to amoisture content not substantially above about 8% by weight prior toimmersion in the liquid, and wherein lthe liquid mixture contains 8 to12% water.

15. A catgut suture conforming to minimum U. S.P tensile strength suturerequirements, said suture comprising a sterile reaction product of astrand of catgut with a solution containing ethylene oxide, the ethyleneoxide being about .01 to 2.5% by liquid volume of the solution, saidreacted catgut having an average breaking time of at least about 3 hoursusing an aqueous papain solution and conditions such that the averagebreaking time for a standard size-0, unbleached, non-hardened,nonboilable gut is 1.8 hours.

16. An interior-ly sterile sealedcontainer containing a tubing liuid anda flexible sterile catgut suture conforming to a minimum U. S. P.tensile strength suture requirements, said suture comprising a sterilereaction product of a strand of catgut with a solution containingethylene oxide, the ethylene oxide being about .01 to 2.5% by liquidvolume of the solution, said reacted catgut having an average breakingtime of at least about 3 hours using an aqueous papain solution andconditions such that the average breaking time for a standard size-0,unbleached, non-hardened, non-boilable gut is 1.8 hours.

17. A container of claim 16 with an average breaking time of at least 6.

18. A container of claim 17 with an average breaking time of at least1l.

19. A cutgut suture conforming to minimum U.S.P tensile strength suturerequirements, said suture comprising u sterile reaction product of astrand of catgut with u solution containing about 2.5 to 20% of waterbased on the weight of the solutions und about 0.01 to 2.5% of ethyleneoxide by liquid volume of the solution, said reacted cuzgut lhaving cmaverage breaking time of or least about 3 hours using un aqueous pupuinsolution and condition such that the average breaking time for astandard size-0, unbleached, non-hardened, non-boilable gul is 1.8hours.

20. An intcrorly sterile sealed container containing a tubing fluid anda flexible sterile cotgut suture conforming to a minimum U.S.P. tensilestrength suture requirements, said suture comprising a sterile reactionproduct of a strand of cotgut with a solution containing about 2.5 to20% of water based on the weight of the solution and about 0.01 to 2.5%of ethylene oxide by liquid volume of the solution, said reacted catguthaving un average breuking time of at least about 3 hours using unaqueous pupain solution and conditions such that the average breakingtime for a standard size-0, unbleuched, non-hardened, non-boilable gutis 1.8 hours.

21. An interiorly sterile sealed container containing a tubing fluid anda flexible sterile catgut suture conforming to minimum U.S.P. tensilestrength suture requirements, said suture comprising a sterile reactionproduct of a strand of catgut with a solution containingabout 2.5 to 20%of water based on the weight of the solution and about 0.01 to 2.5% ofethylene oxide by liquid volume of the solution. I

22. The method of sterilizing a catgut suture which comprises storing anunreacted catgut suture with a tubing fluid, hermctcally sealed in acontainer, said tubing fluid comprising a solution of about .01 to 2.5%

of ethylene oxide by liquid volume and about 2.5 to` 20% of water byweight of the solution and maintaining said sealed container at atemperature in the range of ing to claim 22 wherein the ethylene' oxideis in an amount from about 0.25 to 2.5% by volume.

References Cited in the le of this patent or the original patent UNITEDSTATES PATENTS Davis et al. June 2, 1953 Elson Mar. 13, 1956

